The present application relates to a fluorescence intensity correction method or a fluorescence intensity calculation method, a fluorescence intensity calculation device, and a fluorescence intensity correction program. In more detail, the application relates to a fluorescence intensity correction method for accurately calculating the intensity of fluorescence that is emitted from each of a plurality of fluorescent pigments that are multiply labeled on microparticles, and the like.
In the past, devices (for example, flow cytometers) for measuring the properties of microparticles by labeling microparticles such as cells using fluorescent pigments and measuring the intensity and the pattern of the fluorescence emitted from the fluorescent pigments that are excited by being irradiated by laser light have been used. In recent years, in order to analyze the properties of cells and the like in more detail, multicolor measurement of labeling microparticles using a plurality of fluorescent pigments and measuring the light emitted from each fluorescent pigment by a plurality of light detectors (such as PMTs) having different light-receiving wavelength bands has been performed. In multicolor measurement, detection of fluorescence is performed by selecting an optical filter of the light detector side according to the fluorescence wavelength of the fluorescent pigment that is used.
On the other hand, the fluorescent pigments that are in current usage (for example, FITC, PE (PhycoErythrin), and the like) have wavelength bands that overlap one another in the fluorescence spectrum. Therefore, in a case when multicolor measurement is performed combining such fluorescent pigments, even if the fluorescence emitted from each fluorescent pigment is separated by wavelength bands by an optical filter, fluorescence from fluorescent pigments that are not the target may leak into each light detector. If leaking in of fluorescence occurs, a discrepancy occurs between the fluorescence intensity measured by each light detector and the true fluorescence intensity from the target fluorescent pigments, causing measurement error.
In order to correct for such measurement error, fluorescence correction of deducting the leaked-in fluorescence intensity from the fluorescence intensity measured by the light detectors is performed. Fluorescence correction adds electrical or mathematical correction to pulses in order that the fluorescence intensity measured at the light detectors becomes the true fluorescence intensity from the target fluorescent pigments.
As a method of performing fluorescence correction mathematically, a method of calculating the true fluorescence intensity from the target fluorescent pigments by representing the fluorescence intensity measured at each light detector as a vector and applying an inverse matrix of a leak-in matrix set in advance to the vector is used (refer to FIGS. 9 and 10 and Japanese Unexamined Patent Application Publication No. 2003-83894). The leak-in matrix is created by analyzing the fluorescence wavelength distribution of microparticles in which each florescent pigment is individually singly labeled, and in which the fluorescence wavelength distribution of each fluorescent pigment is arranged as a column vector. The inverse matrix of the leak-in matrix is also referred to as a “correction matrix”. In FIGS. 9 and 10, as an example, a case when 5-color measurement using 5 kinds (FITC, PE, ECD, PC5, PC7) of fluorescent pigments and 5 light detectors is performed is illustrated.